22. A pharmaceutical composition comprising a CRIg variant according to
claim 1, in admixture with a pharmaceutically acceptable excipient.

23. A pharmaceutical composition comprising an immunoadhesin according to
claim 19, in admixture with a pharmaceutically acceptable excipient.

24. A method for the prevention or treatment of a complement-associated
disease or condition, comprising administering to a subject in need of
such treatment a prophlactically or therapeutically effective amount of a
CRIg variant according to claim 1 or an immunoadhesin comprising such
variant.

25. The method of claim 24 wherein said complement-associated disease is
an inflammatory disease or an autoimmune disease.

29. The method of claim 25 wherein said complement-associated disease is a
complenent-associated eye condition.

30. The method of claim 29 wherein said complement-associated eye
condition is selected from the group consisting of all stages of
age-related macular degeneration (AMD), uveitis, diabitic and other
ischemia-related retinopathies, endophthalmitis, and other intraocular
neovascular diseases.

32. The method of claim 29 wherein said complement-associated eye
condition is selected from the group consisting of age-related macular
degeneration (AMD), choroidal neovascularization (CNV), diabetic
retinopathy (DR), and endophthalmitis.

33. The method of claim 32 wherein said AMD is wet AMD.

34. The method of claim 32 wherein said AMD dry or atrophic AMD.

35. The method of claim 24 wherein said subject is a mammal.

36. The method of claim 35 wherein said mammal is a human.

37. A method for inhibition of the production of C3b complement fragment
in a mammal comprising administering to said mammal an effective amount
of a CRIg variant according to claim 1, or an immunoadhesin comprising
said variant.

Description:

[0001]The present application claims the benefit of U.S. Provisional
Patent Application Ser. No. 61/189,653, filed Aug. 20, 2008 and U.S.
Provisional Patent Application Ser. No. 61/050,888, filed May 6, 2008,
the disclosures of which are incorporated hereby by reference in their
entirety.

[0004]The complement system is a complex enzyme cascade made up of a
series of serum glycoproteins that normally exist in inactive, pro-enzyme
form. Three main pathways, the classical, alternative and mannose-binding
lectin pathway, can activate complement, which merge at the level of
where two similar C3 convertases cleave C3 into C3a and C3b.

[0006]Complement consists of over 30 serum proteins that opsonize a wide
variety of pathogens for recognition by complement receptors. Depending
on the initial trigger of the cascade, three pathways can be
distinguished (reviewed by (Walport, N Engl J Med 344, 1058-1066 (2001)).
All three share the common step of activating the central component C3,
but they differ according to the nature of recognition and the initial
biochemical steps leading to C3 activation. The classical pathway is
activated by antibodies bound to the pathogen surface, which in turn bind
the C1q complement component, setting off a serine protease cascade that
ultimately cleaves C3 to its active form, C3b. The lectin pathway is
activated after recognition of carbohydrate motifs by lectin proteins. To
date, three members of this pathway have been identified: the
mannose-binding lectins (MBL), the SIGN-R1 family of lectins and the
ficolins (Pyz et al., Ann Med 38, 242-251 (2006)) Both MBL and ficolins
are associated with serine proteases, which act like C1 in the classical
pathway, activating components C2 and C4 leading to the central C3 step.
The alternative pathway contrasts with both the classical and lectin
pathways in that it is activated due to direct reaction of the internal
C3 ester with recognition motifs on the pathogen surface. Initial C3
binding to an activating surface leads to rapid amplification of C3b
deposition through the action of the alternative pathway proteases Factor
B and Factor D. Importantly, C3b deposited by either the classical or the
lectin pathway also can lead to amplification of C3b deposition through
the actions of Factors B and D. In all three pathways of complement
activation, the pivotal step in opsonization is conversion of the
component C3 to C3b. Cleavage of C3 by enzymes of the complement cascades
exposes the thioester to nucleophilic attack, allowing covalent
attachment of C3b onto antigen surfaces via the thioester domain. This is
the initial step in complement opsonization. Subsequent proteolysis of
the bound C3b produces iC3b, C3c and C3dg, fragments that are recognized
by different receptors (Ross and Medof, Adv Immunol 37, 217-267 (1985)).
This cleavage abolishes the ability of C3b to further amplify C3b
deposition and activate the late components of the complement cascade,
including the membrane attack complex, capable of direct membrane damage.
However, macrophage phagocytic receptors recognize C3b and its fragments
preferentially; due to the versatility of the ester-bond formation,
C3-mediated opsonization is central to pathogen recognition (Holers et
al., Immunol Today 13, 231-236 (1992)), and receptors for the various C3
degradation products therefore play an important role in the host immune
response.

[0007]C3 itself is a complex and flexible protein consisting of 13
distinct domains. The core of the molecule is made up of 8 so-called
macroglobulin (MG) domains, which constitute the tightly packed α
and β chains of C3. Inserted into this structure are CUB (C1r/C1s,
Uegf and Bone mophogenetic protein-1) and TED domains, the latter
containing the thioester bond that allows covalent association of C3b
with pathogen surfaces. The remaining domains contain C3a or act as
linkers and spacers of the core domains. Comparison of C3b and C3c
structures to C3 demonstrate that the molecule undergoes major
conformational rearrangements with each proteolysis, which exposes not
only the TED, but additional new surfaces of the molecule that can
interact with cellular receptors (Janssen and Gros, Mol Immunol 44, 3-10
(2007)).

[0008]Complement C3 Receptors on Phagocytic Cells

[0009]There are three known gene superfamilies of complement receptors:
The short consensus repeat (SCR) modules that code for CR1 and CR2, the
beta-2 integrin family members CR3 and CR4, and the immunoglobulin
Ig-superfamily member CRIg.

[0010]CR1 is a 180-210 kDa glycoprotein consisting of 30 Short Consensus
Repeats (SCRs) and plays a major role in immune complex clearance. SCRs
are modular structures of about 60 amino acids, each with two pairs of
disulfide bonds providing structural rigidity. High affinity binding to
both C3b and C4b occurs through two distinct sites, each composed of 3
SCRs )reviewed by (Krych-Goldberg and Atkinson, Immunol Rev 180, 112-122
(2001)). The structure of the C3b binding site, contained within SCR
15-17 of CR1 (site 2), has been determined by MRI (Smith et al., Cell
108, 769-780 (2002)), revealing that the three modules are in an extended
head-to-tail arrangement with flexibility at the 16-17 junction.
Structure-guided mutagenesis identified a positively charged surface
region on module 15 that is critical for C4b binding. This patch,
together with basic side chains of module 16 exposed on the same face of
CR1, is required for C3b binding. The main function of CR1, first
described as an immune adherence receptor (Rothman et al., J Immunol 115,
1312-1315 (1975)), is to capture ICs on erythrocytes for transport and
clearance by the liver (Taylor et al., Clin Immunol Immunopathol 82,
49-59 (1997)). There is a role in phagocytosis for CR1 on neutrophils,
but not in tissue macrophages (Sengelov et al., J Immunol 153, 804-810
(1994)). In addition to its role in clearance of immune complexes, CR1 is
a potent inhibitor of both classical and alternative pathway activation
through its interaction with the respective convertases (Krych-Goldberg
and Atkinson, 2001, supra; Krych-Goldberg et al., J Biol Chem 274,
31160-31168 (1999)). In the mouse, CR1 and CR2 are two products of the
same gene formed by alternative splicing and are primarily associated
with B-lymphocytes and follicular dendritic cells and function mainly in
regulating B-cell responses (Molina et al., 1996). The mouse functional
equivalent of CR1, Crry, inactivates the classical and alternative
pathway enzymes and acts as an intrinsic regulator of complement
activation rather than as a phagocytic receptor (Molina et al., Proc Natl
Acad Sci USA 93, 3357-3361 (1992)).

[0012]CR3 and CR4 are transmembrane heterodimers composed of an alpha
subunit (CD11b or αM and CD11c or αx, respectively)
and a common beta chain (CD18 or β2), and are involved in
adhesion to extracellular matrix and to other cells as well as in
recognition of iC3b. They belong to the integrin family and perform
functions not only in phagocytosis, but also in leukocyte trafficking and
migration, synapse formation and costimulation (reviewed by (Ross, Adv
Immunol 37, 217-267 (2000)). Integrin adhesiveness is regulated through a
process called inside-out signaling, transforming the integrins from a
low- to a high-affinity binding state (Liddington and Ginsberg, J Cell
Biol 158, 833-839 (2002)). In addition, ligand binding transduces signals
from the extracellular domain to the cytoplasm. The binding sites of iC3b
have been mapped to several domains on the alpha chain of CR3 and CR4
(Diamond et al., J Cell Biol 120, 1031-1043 (1993); Li and Zhang, J Biol
Chem 278, 34395-34402 (2003); Xiong and Zhang, J Biol Chem 278,
34395-34402 (2001)). The multiple ligands for CR3: iC3b, beta-glucan and
ICAM-1, seem to bind to partially overlapping sites contained within the
I domain of CD11b (Balsam et al., 1998; Diamond et al., 1990; Zhang and
Plow, 1996). Its specific recognition of the proteolytically inactivated
form of C3b, iC3b, is predicted based on structural studies that locate
the CR3 binding sites to residues that become exposed upon unfolding of
the CUB domain in C3b (Nishida et al., Proc Natl Acad Sci U S A 103,
19737-19742 (2006)), which occurs upon α' chain cleavage by the
complement regulatory protease, Factor I.

[0013]CRIg is a macrophage associated receptor with homology to A33
antigen and JAM1 that is required for the clearance of pathogens from the
blood stream. A human CRIg protein was first cloned from a human fetal
cDNA library using degenerate primers recognizing conserved Ig domains of
human JAM1. Sequencing of several clones revealed an open reading frame
of 400 amino acids. Blast searches confirmed similarity to Z39Ig, a type
1 transmembrane protein (Langnaese et al., Biochim Biophys Acta 1492
(2000) 522-525). The extracellular region of this molecule was found to
consist of two Ig-like domains, comprising an N-terminal V-set domain and
a C-terminal C2-set domain. The novel human protein was originally
designated as a "single transmembrane Ig superfamily member macrophage
associated" (huSTIgMA). (huSTIgMA). Subsequently, using 3' and 5'
primers, a splice variant of huSTIgMA was cloned, which lacks the
membrane proximal IgC domain and is 50 amino acids shorter. Accordingly,
the shorter splice variant of this human protein was designated
huSTIgMAshort. The amino acid sequence of huSTIgMA (referred to as
PRO362) and the encoding polynucleotide sequence are disclosed in U.S.
Pat. No. 6,410,708, issued Jun. 25, 2002. In addition, both huSTIgMA and
huSTIgMAshort, along with the murine STIgMA (muSTIgMA) protein and
nucleic acid sequences, are disclosed in PCT Publication WO 2004031105,
published Apr. 15, 2004.

[0014]The crystal structure of CRIg and a C3b:CRIg complex is disclosed in
U.S. Application Publication No. 2008/0045697, published Feb. 21, 2008.

[0015]The Kupffer cells (KCs), residing within the lumen of the liver
sinusoids, form the largest population of macrophages in the body.
Although KCs have markers in common with other tissue resident
macrophages, they perform specialized functions geared towards efficient
clearance of gut-derived bacteria, microbial debris, bacterial
endotoxins, immune complexes and dead cells present in portal vein blood
draining from the microvascular system of the digestive tract (Bilzer et
al., Liver Int 26, 1175-1186 (2006)). Efficient binding of pathogens to
the KC surface is a crucial step in the first-line immune defense against
pathogens (Benacerraf et al., J Exp Med 110, 27-48 (1959)). A central
role for KCs in the rapid clearance of pathogens from the circulation is
illustrated by the significantly increased mortality in mice depleted of
KCs (Hirakata et al., Infect Immun 59, 289-294 (1991)). The
identification of CRIg further stresses the critical role of complement
and KCs in the first line immune defense against circulating pathogens.

[0016]The only complement C3 receptors identified on mouse KCs are CRIg
and CR3 (Helmy et al., Cell 124, 915-927 (2006)), while human KCs show
additional expression of CR1 and CR4 (Hinglais et al., 1989). Both CRIg
and CR3 on KCs contribute to binding to iC3b opsonized particles in vitro
(Helmy et al., Lab Invest 61, 509-514 (2006)). In vivo, a role of
KC-expressed CR3 in the binding to iC3b-coated pathogens is less clear.
CR3 has been proposed to contribute to clearance of pathogens indirectly
via recruitment of neutrophils and interaction with neutrophil-expressed
ICAMI (Conlan and North, Exp Med 179, 2,59-268 (1994); Ebe et al., Pathol
Int 49, 519-532 (1999); Gregory et al., J Immunol 157, 2514-2520 (1996);
Gregory and Wing, J Leukoc Biol 72, 239-248 (2002); Rogers and Unanue,
Infect Immun 61, 5090-5096 (1993)). In contrast, CRIg performs a direct
role by capturing pathogens that transit through the liver sinusoidal
lumen (Helmy et al., 2006, supra). A difference in the biology of CRIg vs
CR3 is in part reflected by difference in binding characteristics of
these two receptors. CRIg expressed on KCs constitutively binds to
monomeric C3 fragments whereas CR3 only binds to iC3b-opsonized particles
(Helmy et al., 2006, supra). The capacity of CRIg to efficiently capture
monomeric C3b and iC3b as well as C3b/iC3b-coated particles reflects the
increased avidity created by a multivalent interaction between CRIg
molecules concentrated at the tip of membrane extensions of macrophages
(Helmy et al., 2006, supra) and multimers of C3b and iC3b present on the
pathogen surface. While CR3 only binds iC3b-coated particles, CRIg
additionally bind to C3b, the first C3 cleavage product formed on
serum-opsonized pathogens (Croize et al., Infect Immun 61, 5134-5139
(1993)). Since a large number of C3b molecules bound to the pathogen
surface are protected from cleavage by factor H and I (Gordon et al., J
Infect Dis 157, 697-704 (1988)), recognition of C3b ligands by CRIg
ensures rapid binding and clearance. Thus, while both CRIg and CR3 are
expressed on KCs, they show different ligand specificity, distinct
binding properties and distinct kinetics of pathogen clearance.

[0018]As discussed above, CRIg is a recently discovered complement C3
receptor expressed on a subpopulation of tissue resident macrophages.
Next to functioning as a complement receptor for C3 proteins, the
extracellular IgV domain of CRIg selectively inhibits the alternative
pathway of complement by binding to C3b and inhibiting proteolytic
activation of C3 and C5. However, CRIg binding affinity for the
convertase subunit C3b is low (IC50>1 μM) requiring a relatively
high concentration of protein to reach near complete complement
inhibition. Accordingly, there is a need for CRIg polypeptides with
improved therapeutic efficacy. The present invention provides such
polypeptides.

SUMMARY OF THE INVENTION

[0019]The present invention is based, at least in part, on the
construction of a CRIg variant with enhanced binding affinity. A CRIg-ECD
protein with combined amino acid substitutions Q64R and M86Y showed a 30
fold increased binding affinity and a 7 fold improved complement
inhibitory activity over the wildtype CRIg variant. In addition,
treatment with the affinity-improved CRIg fusion protein in a mouse model
of arthritis resulted in a significant reduction in clinical scores
compared to treatment with a wild-type CRIg protein

[0020]Accordingly, the present invention concerns CRIg variants.

[0021]In one aspect, the invention concerns a CRIg variant comprising an
amino acid substitution in a region selected from the group consisting of
E8-K15, R41-T47, S54-Q64, E85-Q99, and Q105-K111 of the amino acid
sequence of SEQ ID NO: 2.

[0022]In one embodiment, the variant selectively binds to C3b over C3, or
a fragment thereof.

[0023]In another embodiment, the variant has increased binding affinity to
C3b over native sequence human CRIg of SEQ ID NO: 2, where the binding
affinity may, for example, be increased by at lest 2 fold, or by at least
3 fold, or by at least 4 fold, or by at least 5 fold, or by at least 6
fold, or by at least 7 fold, or by at least 9 fold, or by at least 10
fold, or by at least 15 fold, or by at least 20 fold, or by at least 30
fold, or by at least 40 fold, or by at least 50 fold, or by at least 70
fold, or by at least 80 fold, or by at least 90 fold, or by at least 100
fold.

[0024]In yet another embodiment, the variant is a more potent inhibitor of
the alternative complement pathway than native sequence human CRIg of SEQ
ID NO: 2.

[0025]In a further embodiment, the variant comprises an amino acid
substitution at one or more amino acid positions selected from the group
consisting of positions 8, 14, 18, 42, 44, 45, 60, 64, 86, 99, 105, and
110 in the amino acid sequence of SEQ ID NO: 2.

[0026]In a still further embodiment, the variant comprises an amino acid
substitution at one or more of amino acid positions 60, 64, 86, 99, 105
and 110 in the amino acid sequence of SEQ ID NO: 2.

[0035]In a further aspect, the invention concerns a pharmaceutical
composition comprising a CRIg variant or a chimeric molecule, e.g. an
immunoadhesin of the present invention, in admixture with a
pharmaceutically acceptable excipient.

[0036]In a still further aspect, the invention concerns a method for the
prevention or treatment of a complement-associated disease or condition,
comprising administering to a subject in need of such treatment a
prophylactically or therapeutically effective amount of a CRIg variant or
a chimeric molecule, such as an immunoadhesin, comprising such variant.

[0037]In one embodiment, the complement-associated disease is an
inflammatory disease or an autoimmune disease.

[0041]In another preferred embodiment, the complement-associated disease
is a complement-associated eye condition.

[0042]In a further embodiment, the complement-associated eye condition is
selected from the group consisting of all stages of age-related macular
degeneration (AMD), uveitis, diabetic and other ischemia-related
retinopathies, endophthalmitis, and other intraocular neovascular
diseases.

[0043]In a still further embodiment, the intraocular neovascular disease
is selected from the group consisting of diabetic macular edema,
pathological myopia, von Hippel-Lindau disease, histoplasmosis of the
eye, Central Retinal Vein Occlusion (CRVO), corneal neovascularization,
and retinal neovascularization.

[0044]In yet another embodiment, the complement-associated eye condition
is selected from the group consisting of age-related macular degeneration
(AMD), choroidal neovascularization (CNV), diabetic retinopathy (DR), and
endophthalmitis, where AMD includes both wet and dry or atrophic AMD.

[0045]In one embodiment, the patient is a mammal, preferable a human.

[0046]In another aspect, the invention concerns a method for inhibition of
the production of C3b complement fragment in a mammal comprising
administering to said mammal an effective amount of a CRIg variant of the
present invention, or an immunoadhesin comprising such variant.

[0047]In yet another aspect, the invention concerns a method for selective
inhibition of the alternative complement pathway in a mammal, comprising
administering to said mammal an effective amount of a CRIg variant of the
present invention, or an immunoadhesin comprising such variant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048]FIGS. 1A-1B show the nucleotide and amino acid sequences of the
399-amino acid full-length long form of native human CRIg (huCRIg, SEQ ID
NOS: 1 and 2, respectively).

[0049]FIGS. 2A-2B show the nucleotide and amino acid sequences of the
305-amino acid short form of native human CRIg (huCRIg-short, SEQ ID NOS:
3 and 4, respectively).

[0051]FIG. 4: Activity of CRIg mutants in binding assay and inhibition
assay. Binding affinity for CRIg was measured as competitive displacement
of C3b (A), and the biological activity was measured by a hemolysis
inhibition assay. PUR10680 was wild-type control (red), RIL41 (blue) and
RL41 (green) were two mutants (B). (C) Stepwise optimization of the CRIg
binding interface.

[0057]Table 1: Phage libraries. Five soft-randomized libraries were
designed to cover the contact area between CRIg and C3b.

[0058]Table 2: Step-wise generation of higher affinity CRIg my phage
display. Selected mutants of CRIg anti-C3b from the five soft-randomized
libraries. Each panel shows clones that were selected from each library
based on binding affinity to C3b. The sequence is denoted by the
single-letter amino acid code. Each panel compares the individual mutants
with the consensus and parent wild-type (WT) sequences. Residues are
colored accordingly: blue--soft randomized position; gray--not
randomized; yellow - the selected residues, which are different from
wild-type (WT). Table 2 discloses SEQ ID NOS 21-63 and 63-67,
respectively, in order of appearance.

[0059]Table 3: Comparison of binding affinities, determined by competitive
ELISA, and in vivo hemolysis inhibition for selected mutants. Mutants
with a greater than 5 fold increased in binding affinity or in vivo
potency are shaded yellow.

[0060]Table 4: Comparison of binding affinity and in vivo hemolysis
inhibition for second generation mutants (parent sequences shown in
gray). Mutants with a greater than 5 fold increase over the parent mutant
in binding affinity are highlighted in blue, mutants with a greater than
90 fold increase in binding affinity are highlighted in yellow.
Similarly, mutants with greater in vivo potency than parent sequences are
highlighted in orange.

DETAILED DESCRIPTION OF THE INVENTION

[0061]I. Definitions

[0062]The terms "CRIg," "PRO362," "JAM4," and "STIgMA" are used
interchangeably, and refer to native sequence and variant CRIg
polypeptides.

[0063]A "native sequence" CRIg, is a polypeptide having the same amino
acid sequence as a CRIg polypeptide derived from nature, regardless of
its mode of preparation. Thus, native sequence CRIg can be isolated from
nature or can be produced by recombinant and/or synthetic means. The term
"native sequence CRIg", specifically encompasses naturally-occurring
truncated or secreted forms of CRIg (e.g., an extracellular domain
sequence), naturally-occurring variant forms (e.g., alternatively spliced
forms) and naturally-occurring allelic variants of CRIg.

[0064]Native sequence CRIg polypeptides specifically include the
full-length 399 amino acids long human CRIg polypeptide of SEQ ID NO: 2
(huCRIg, shown in FIGS. 1A and 1B), with or without an N-terminal signal
sequence, with or without the initiating methionine at position 1, and
with or without any or all of the transmembrane domain at about amino
acid positions 277 to 307 of SEQ ID NO: 2. In a further embodiment, the
native sequence CRIg polypeptide is the 305-amino acid, short form of
human CRIg (huCRIg-short, SEQ ID NO: 4, shown in FIGS. 2A and 2B), with
or without an N-terminal signal sequence, with or without the initiating
methionine at position 1, and with or without any or all of the
transmembrane domain at about positions 183 to 213 of SEQ ID NO: 4. In a
different embodiment, the native sequence CRIg polypeptide is a 280 amino
acids long, full-length murine CRIg polypeptide of SEQ ID NO: 6 (muCRIg,
shown in FIGS. 3A-3C), with or without an N-terminal signal sequence,
with or without the initiating methionine at position 1, and with or
without any or all of the transmembrane domain at about amino acid
positions 181 to 211 of SEQ ID NO: 6. CRIg polypeptides of other
non-human animals, including higher primates and mammals, are
specifically included within this definition.

[0065]The CRIg "extracellular domain" or "ECD" refers to a form of the
CRIg polypeptide, which is essentially free of the transmembrane and
cytoplasmic domains of the respective full length molecules. Ordinarily,
the CRIg ECD will have less than 1% of such transmembrane and/or
cytoplasmic domains and preferably, will have less than 0.5% of such
domains. CRIg ECD may comprise amino acid residues 1 or about 21 to X of
SEQ ID NO: 2, 4, or 6, where X is any amino acid from about 271 to 281 in
SEQ ID NO: 2, any amino acid from about 178 to 186 in SEQ ID NO: 4, and
any amino acid from about 176 to 184 in SEQ ID NO: 6.

[0066]The term "CRIg variant," as used herein, means an active CRIg
polypeptide as defined below having at least about 80% amino acid
sequence identity to a native sequence CRIg polypeptide, including,
without limitation, the full-length huCRIg (SEQ ID NO: 2), huCRIg-short
(SEQ ID NO: 4), and muCRIg (SEQ ID NO: 6), each with or without the
N-terminal initiating methionine, with or without the N-terminal signal
sequence, with or without all or part of the transmembrane domain and
with or without the intracellular domain. In a particular embodiment, the
CRIg variant has at least about 80% amino acid sequence homology with the
mature, full-length polypeptide from within the sequence of the sequence
of SEQ ID NO: 2. In another embodiment, the CRIg variant has at -least
about 80% amino acid sequence homology with the mature, full-length
polypeptide from within the sequence of SEQ ID NO: 4. In yet another
embodiment, the CRIg variant has at least about 80% amino acid sequence
homology with the mature, full-length polypeptide from within the
sequence of SEQ ID NO: 6. Ordinarily, a CRIg variant will have at least
about 80% amino acid sequence identity, or at least about 85% amino acid
sequence identity, or at least about 90% amino acid sequence identity, or
at least about 95% amino acid sequence identity, or at least about 98%
amino acid sequence identity, or at least about 99% amino acid sequence
identity with the mature amino acid sequence from within SEQ ID NO: 2, 4,
or 6. Throughout the description, including the examples, the term
"wild-type" or "WT" refers to the mature full-length short form of human
CRIg (CRIg(S)) (SEQ ID NO: 4), and the numbering of amino acid residues
in the CRIg variants refers to the sequence of SEQ ID NO: 4

[0067]The CRIg variants of the present invention are CRIg agonists, as
hereinafter defined. In particular, the CRIg variants herein maintain
selective binding to C3b over C3, where "selective binding" is used to
refer to binding to C3b and a lack of binding to C3. In addition, in a
preferred embodiment, the CRIg variants of the present invention have
increased binding affinity to C3b relative to a native sequence CRIg
polypeptide, such as the human long form of CRIg (SEQ ID NO: 2). In
various embodiments, the increase in binding affinity is at least about 2
fold, or at least about 3 fold, or at least about 4 fold, or at least
about 5 fold, or at least about 6 fold, or at least about 7 fold, or at
least about 8 fold, or at least about 9 fold, or at least about 10 fold,
or at least about 15 fold, or at least about 20 fold, or at least about
25 fold, or at least about 30 fold, or at least about 35 fold, or at
least about 40 fold, or at least about 45 fold, or at least about 50
fold, or at least about 55 fold, or at least about 60 fold, or at least
about 65 fold, or at least about 70 fold, or at least about 75 fold, or
at least about 80 fold, or at least about 85 fold, or at least about 90
fold, or at least about 95 fold, or at least about 100 fold, relative to
the native sequence human CRIg polypeptide of SEQ ID NO: 2. In other
embodiments, the increase in binding affinity to C3b relative to the
native sequence human CRIg polypeptide of SEQ ID NO: 2 is about 5-10
fold, or about 5-15 fold, or about 5-20 fold, or about 5-25 fold, or
about 5-25 fold, or about 5-30 fold, or about 5-35 fold, or about 5-40
fold, or about 5-45 fold, or about 5-50 fold, or about 5-55 fold, or
about 5-60 fold, or about 5-65 fold, or about 5-70 fold, or about 5-75
fold, or about 5-80 fold, or about 5-85 fold, or about 5-90 fold, or
about 5-95 fold, or about 5-100 fold.

[0068]"Percent (%) amino acid sequence identity" with respect to the CRIg
variants herein is defined as the percentage of amino acid residues in a
CRIg variant sequence that are identical with the amino acid residues in
the native CRIg sequence to which they are compared, after aligning the
sequences and introducing gaps, if necessary, to achieve the maximum
percent sequence identity, and not considering any conservative
substitutions as part of the sequence identity. For sequences that differ
in length, percent sequence identity is determined relative to the longer
sequence, along the full length of the longer sequences. Alignment for
purposes of determining percent amino acid sequence identity can be
achieved in various ways that are within the skill in the art, for
instance, using publicly available computer software such as BLAST,
BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art
can determine appropriate parameters for measuring alignment, including
any algorithms needed to achieve maximal alignment over the full length
of the sequences being compared. Sequence identity is then calculated
relative to the longer sequence, i.e. even if a shorter sequence shows
100% sequence identity with a portion of a longer sequence, the overall
sequence identity will be less than 100%.

[0069]"Percent (%) nucleic acid sequence identity" with respect to the
CRIg variant encoding sequences identified herein is defined as the
percentage of nucleotides in a candidate sequence that are identical with
the nucleotides in the CRIg variant encoding sequence, respectively,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity. Alignment for purposes of
determining percent nucleic acid sequence identity can be achieved in
various ways that are within the skill in the art, for instance, using
publicly available computer software such as BLAST, BLAST-2, ALIGN or
Megalign (DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any algorithms
needed to achieve maximal alignment over the full length of the sequences
being compared. Sequence identity is then calculated relative to the
longer sequence, i.e. even if a shorter sequence shows 100% sequence
identity with a portion of a longer sequence, the overall sequence
identity will be less than 100%.

[0070]Included in the definition of a CRIg variant are all amino acid
sequence variants, as hereinabove defined, regardless of their mode of
identification or preparation. Specifically included herein are variants
that have been modified by substitution, chemically, enzymatically, or by
other appropriate means with a moiety other than a naturally occurring
amino acid, as long as they retain a qualitative biological property of a
native sequence CRIg. Exemplary non-naturally occurring amino acid
substitution include those described herein below.

[0071]Amino acid residues are classified into four major groups:

[0072]Acidic: The residue has a negative charge due to loss of H ion at
physiological pH and the residue is attracted by aqueous solution so as
to seek the surface positions in the conformation of a peptide in which
it is contained when the peptide is in aqueous solution.

[0073]Basic: The residue has a positive charge due to association with H
ion at physiological pH and the residue is attracted by aqueous solution
so as to seek the surface positions in the conformation of a peptide in
which it is contained when the peptide is in aqueous medium at
physiological pH.

[0074]Neutral/non-polar: The residues are not charged at physiological pH
and the residue is repelled by aqueous solution so as to seek the inner
positions in the conformation of a peptide in which it is contained when
the peptide is in aqueous medium. These residues are also designated
"hydrophobic residues."

[0075]Neutral/polar: The residues are not charged at physiological pH, but
the residue is attracted by aqueous solution so as to seek the outer
positions in the conformation of a peptide in which it is contained when
the peptide is in aqueous medium.

[0076]Amino acid residues can be further classified as cyclic or
non-cyclic, aromatic or non aromatic with respect to their side chain
groups these designations being commonplace to the skilled artisan.

[0078]As used herein, the term "immunoadhesin" designates antibody-like
molecules which combine the binding specificity of a heterologous protein
(an "adhesin") with the effector functions of immunoglobulin constant
domains. Structurally, the immunoadhesins comprise a fusion of an amino
acid sequence with the desired binding specificity which is other than
the antigen recognition and binding site of an antibody (i.e., is
"heterologous"), and an immunoglobulin constant domain sequence. The
adhesin part of an immunoadhesin molecule typically is a contiguous amino
acid sequence comprising at least the binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the immunoadhesin
may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or
IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
"Treatment" is an intervention performed with the intention of preventing
the development or altering the pathology of a disorder. Accordingly,
"treatment" refers to both therapeutic treatment and prophylactic or
preventative measures. Those in need of treatment include those already
with the disorder as well as those in which the disorder is to be
prevented.

[0079]"Ameliorate" as used herein, is defined herein as to make better or
improve.

[0080]The term "mammal" as used herein refers to any animal classified as
a mammal, including, without limitation, humans, non-human primates,
domestic and farm animals, and zoo, sports or pet animals such horses,
pigs, cattle, dogs, cats and ferrets, etc. In a preferred embodiment of
the invention, the mammal is a higher primate, most preferably human.

[0081]The term "complement-associated disease" is used herein in the
broadest sense and includes all diseases and pathological conditions the
pathogenesis of which involves abnormalities of the activation of the
complement system, such as, for example, complement deficiencies. The
term specifically include diseases and pathological conditions that
benefit from the inhibition of C3 convertase. The term additionally
includes diseases and pathological conditions that benefit from
inhibition, including selective inhibition, of the alternative complement
pathway. Complement-associated diseases include, without limitation,
inflammatory diseases and autoimmune diseases, such as, for example,
rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS),
remote tissue injury after ischemia and reperfusion, complement
activation during cardiopulmonary bypass surgery, dermatomyositis,
pemphigus, lupus nephritis and resultant glomerulonephritis and
vasculitis, cardiopulmonary bypass, cardioplegia-induced coronary
endothelial dysfunction, type II membranoproliferative
glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulemia,
antiphospholipid syndrome, age-related macular degeneration, uveitis,
diabetic retinopathy, allo-transplantation, hyperacute rejection,
hemodialysis, chronic occlusive pulmonary distress syndrome (COPD),
asthma, and aspiration pneumonia. In a preferred embodiment, the
"complement-associated disease" is a disease in which the alternative
pathway of complement plays a prominent role, including rheumatoid
arthritis (RA), complement-associated eye conditions, such as age-related
macular degeneration, anti-phospholipid syndrome, intestinal and renal
ischemia-reperfusion injury, and type II membranoproliferative
glomerulonephritis.

[0082]The term "complement-associated eye condition" is used herein in the
broadest sense and includes all eye conditions and diseases the pathology
of which involves complement, including the classical and the alternative
pathways, and in particular the alternative pathway of complement.
Specifically included within this group are all eye conditions and
diseases the associated with the alternative pathway, the occurrence,
development, or progression of which can be controlled by the inhibition
of the alternative pathway. Complement-associated eye conditions include,
without limitation, macular degenerative diseases, such as all stages of
age-related macular degeneration (AMD), including dry and wet
(non-exudative and exudative) forms, choroidal neovascularization (CNV),
uveitis, diabetic and other ischemia-related retinopathies,
endophthalmitis, and other intraocular neovascular diseases, such as
diabetic macular edema, pathological myopia, von Hippel-Lindau disease,
histoplasmosis of the eye, Central Retinal Vein Occlusion (CRVO), corneal
neovascularization, and retinal neovascularization. A preferred group of
complement-associated eye conditions includes age-related macular
degeneration (AMD), including non-exudative (wet) and exudative (dry or
atrophic) AMD, choroidal neovascularization (CNV), diabetic retinopathy
(DR), and endophthalmitis.

[0083]The term "inflammatory disease" and "inflammatory disorder" are used
interchangeably and mean a disease or disorder in which a component of
the immune system of a mammal causes, mediates or otherwise contributes
to an inflammatory response contributing to morbidity in the mammal. Also
included are diseases in which reduction of the inflammatory response has
an ameliorative effect on progression of the disease. Included within
this term are immune-mediated inflammatory diseases, including autoimmune
diseases.

[0084]The term "T-cell mediated" disease means a disease in which T cells
directly or indirectly mediate or otherwise.contribute to morbidity in a
mammal. The T cell mediated disease may be associated with cell mediated
effects, lymphokine mediated effects, etc. and even effects associated
with B cells if the B cells are stimulated, for example, by the
lymphokines secreted by T cells.

[0086]Administration "in combination with" one or more further therapeutic
agents includes simultaneous (concurrent) and consecutive administration
in any order.

[0087]"Active" or "activity" in the context of variants of the CRIg
polypeptides of the invention refers to form(s) of such polypeptides
which retain the biological and/or immunological activities of a native
or naturally-occurring polypeptide of the invention. A preferred
biological activity is the ability to bind C3b, and/or to affect
complement or complement activation, in particular to inhibit the
alternative complement pathway and/or C3 convertase. Inhibition of C3
convertase can, for example, be measured by measuring the inhibition of
C3 turnover in normal serum during collagen- or antibody-induced
arthritis, or inhibition of C3 deposition is arthritic joints.
"Biological activity" in the context of a polypeptide that mimics CRIg
biological activity refers, in part, to the ability of such molecules to
bind C3b and/or to affect complement or complement activation, in
particular, to inhibit the alternative complement pathway and/or C3
convertase.

[0088]The term CRIg "agonist" is used in the broadest sense, and includes
any molecule that mimics a qualitative biological activity (as
hereinabove defined) of a native sequence CRIg polypeptide.

[0089]"Operably linked" refers to juxtaposition such that the normal
function of the components can be performed. Thus, a coding sequence
"operably linked" to control sequences refers to a configuration wherein
the coding sequence can be expressed under the control of these sequences
and wherein the DNA sequences being linked are contiguous and, in the
case of a secretory leader, contiguous and in reading phase. For example,
DNA for a presequence or secretory leader is operably linked to DNA for a
polypeptide if it is expressed as a preprotein that participates in the
secretion.of the polypeptide; a promoter or enhancer is operably linked
to a coding sequence if it affects the transcription of the sequence; or
a ribosome binding site is operably linked to a coding sequence if it is
positioned so as to facilitate translation. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not exist,
then synthetic oligonucleotide adaptors or linkers are used in accord
with conventional practice.

[0090]"Control sequences" refer to DNA sequences necessary for the
expression of an operably linked coding sequence in a particular host
organism. The control sequences that are suitable for prokaryotes, for
example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells are known to utilize promoters,
polyadenylation signals, and enhancers.

[0091]"Expression system" refers to DNA sequences containing a desired
coding sequence and control sequences in operable linkage, so that hosts
transformed with these sequences are capable of producing the encoded
proteins. To effect transformation, the expression system may be included
on a vector; however, the relevant DNA may then also be integrated into
the host chromosome.

[0092]As used herein, "cell," "cell line," and "cell culture" are used
interchangeably and all such designations include progeny. Thus,
"transformants" or "transformed cells" includes the primary subject cell
and cultures derived therefrom without regard for the number of
transfers. It is also understood that all progeny may not be precisely
identical in DNA content because deliberate or inadvertent mutations may
occur. Mutant progeny that have the same functionality as screened for in
the originally transformed cell are included. Where distinct designations
are intended, it will be clear from the context.

[0093]"Plasmids" are designated by a lower case "p" preceded and/or
followed by capital letters and/or numbers. The starting plasmids herein
are commercially available, are publicly available on an unrestricted
basis, or can be constructed from such available plasmids in accord with
published procedures. In addition, other equivalent plasmids are known in
the art and will be apparent to the ordinary artisan.

[0094]A "phage display library" is a protein expression library that
expresses a collection of cloned protein sequences as fusions with a
phage coat protein. Thus, the phrase "phage display library" refers
herein to a collection of phage (e.g., filamentous phage) wherein the
phage express an external (typically heterologous) protein. The external
protein is free to. interact with (bind to) other moieties with which the
phage are contacted. Each phage displaying an external protein is a
"member" of the phage display library.

[0095]The term "filamentous phage" refers to a viral particle capable of
displaying a heterogenous polypeptide on its surface, and includes,
without limitation, f1, fd, Pf1, and M13. The filamentous phage may
contain a selectable marker such as tetracycline (e.g., "fd-tet").
Various filamentous phage display systems are well known to those of
skill in the art (see, e.g., Zacher et al., Gene, 9:127-140 (1980), Smith
et al., Science, 228:1315-1317 (1985); and Parmley and Smith, Gene,
73:305-318 (1988)).

[0096]The term "panning" is used to refer to the multiple rounds of
screening process in identification and isolation of phages carrying
compounds, such as antibodies, with high affinity and specificity to a
target.

[0097]The phrase "conserved amino acid residues" is used to refer to amino
acid residues that are identical between two or more amino acid sequences
aligned with each other.

II. DETAILED DESCRIPTION

[0098]Complement is an important component of the innate and adaptive
immune response, yet complement split products generated through
activation of each of the three complement pathways (classical,
alternative, and lectin) can cause inflammation and tissue destruction.
Thus, uncontrolled complement activation due to the lack of appropriate
complement regulation has been associated with various chronic
inflammatory diseases. Dominant in this inflammatory cascade are the
complement split products C3a and C5a that function as chemoattractant
and activators of neutrophils and inflammatory macrophages via the C3a
and C5a receptors (Mollnes, T. E., W. C. Song, and J. D. Lambris. 2002.
Complement in inflammatory tissue damage and disease. Trends Immunol.
23:61-64.

[0099]CRIg is a recently discovered complement receptor, which is
expressed on a subpopulation of tissue resident macrophages. As a
functional receptor, the extracellular IgV domain of CRIg is a selective
inhibitor of the alternative pathway of complement (Wiesmann et al.,
Nature, 444(7116):217-20, 2006). A soluble form of CRIg has been shown to
reverse inflammation and bone loss in experimental models of arthritis by
inhibiting the alternative pathway of complement in the joint. It has
also been shown that the alternative pathway of complement is not only
required for disease induction, but also disease progression. Thus,
inhibition of the alternative pathway by CRIg constitutes a promising
therapeutic avenue for the prevention and treatment of diseases and
disorders the pathogenesis of which involves the alternative pathway of
complement. For further details see, e.g. Helmy et al., Cell,
125(1):29-32 2006) and Katschke et al., J. Exp Med 204(6):1319-1325
(2007).

[0100]However, CRIg affinity for the convertase subunit C3b is low
(micromolar range). In order to generate a more potent inhibitor to
develop a therapeutic reagent, the crystal structure of CRIg in complex
with C3b was used as a guide and we employed phage display technology to
generate CRIg variants with improved binding affinity for C3b.

[0103]As described in greater detail in the Example, phage display of
protein or peptide libraries offers a useful methodology for the
selection of CRIg variants with improved binding affinity for C3b and/or
other improved properties, such as enhanced biological activity (Smith,
G. P., (1991) Curr. Opin. Biotechnol. 2:668-673). High affinity proteins,
displayed in a monovalent fashion as fusions with the M13 gene III coat
protein (Clackson, T., (1994) et al., Trends Biotechnol. 12:173-183), can
be identified by cloning and sequencing the corresponding DNA packaged in
the phagemid particles after a number of rounds of binding selection.

[0104]Affinity maturation using phage display has been described, for
example, in Lowman et al., Biochemistry 30(45): 10832-10838 (1991), see
also Hawkins et al, J. Mol Biol.254: 889-896 (1992), and in the Example
below. While not strictly limited to the following description, this
process can be described briefly as: several sites within a predetermined
region are mutated to generate all possible amino acid substitutions at
each site. The antibody mutants thus generated are displayed in a
monovalent fashion from filamentous phage particles as fusions to the
gene III product of M13 packaged within each particle. The phage
expressing the various mutants can be cycled through rounds of binding
selection, followed by isolation and sequencing of those mutants which
display high affinity. The method is also described in U.S. Pat. No.
5,750,373, issued May 12, 1998.

[0105]A modified procedure involving pooled affinity display is described
in Cunningham, B. C. et al, EMBO J. 13(11), 2508-2515 (1994). The method
provides a method for selecting novel binding polypeptides comprising: a)
constructing a replicable expression vector comprising a first gene
encoding a polypeptide, a second gene encoding at least a portion of a
natural or wild-type phage coat protein wherein the first and second
genes are heterologous, and a transcription regulatory element operably
linked to the first and second genes, thereby forming a gene fusion
encoding a fusion protein; b) mutating the vector at one or more selected
positions within the first gene thereby forming a family of related
plasmids; c) transforming suitable host cells with the plasmids; d)
infecting the transformed host cells with a helper phage having a gene
encoding the phage coat protein; e) culturing the transformed infected
host cells under conditions suitable for forming recombinant phagemid
particles containing at least a portion of the plasmid and capable of
transforming the host, the conditions adjusted so that no more than a
minor amount of phagemid particles display more than one copy of the
fusion protein on the surface of the particle; f) contacting the phagemid
particles with a target molecule so that at least a portion of the
phagemid particles bind to the target molecule; and g) separating the
phagemid particles that bind from those that do not. Preferably, the
method further comprises transforming suitable host cells with
recombinant phagemid particles that bind to the target molecule and
repeating steps d) through g) one or more times.

[0106]It is noted that, while the CRIg variants of the present invention
have been identified using phage display, other techniques and other
display techniques can also be used to identify CRIg variants with
improved properties, including affinity matured CRIg variants.

[0107]The affinity matured CRIg variants of the present invention were
designed to cover the contact area between CRIg and C3b, which was
identified using the crystal structure of a CRIg and C3b:CRIg complex
disclosed in U.S. application publication no. 20080045697. I particular,
as shown in Table 1, libraries 1-5 were designed to cover residues
E8-K15, R41-T47, S54-Q64, E85-Q99, and Q105-K111, respectively, of the
native sequence full-length CRIg molecule of SEQ ID NO: 2.

[0108]In one embodiment, the CRIg variants herein contain an amino acid
substitution at one or more amino acid positions selected from the group
consisting of positions 8, 14, 18, 42, 44, 45, 60, 64, 86, 99, 105, and
110 in the amino acid sequence of SEQ ID NO: 2.

[0109]Representative CRIg variants herein are set forth in Table 3.

[0110]Preferably, the substitution is at one or more of amino acid
positions 60, 64, 86, 99, 105 and 110 of the amino acid sequence of
full-length native CRIg of SEQ ID NO: 2.

[0115]Variants which contain one or more of the mutations listed above or
in Tables 3 and 4 but otherwise retain the native CRIg sequence of SEQ ID
NO: 2 are specifically included herein. Such variants will be designated
herein by listing the particular mutation followed by "CRIg." Thus for
example, a variant which differs from native sequence CRIg of SEQ ID NO:
2 only by the mutation E8W will be designated as "E8W CRIg," a variant
which differs from native sequence CRIg of SEQ ID NO: 2 only by the
mutations Q60I/Q64R/M86Y will be designated as "Q60I/Q64R/M86Y CRIg,"
etc.

[0116]Preparation of CRIg Variants

[0117]Various techniques are available which may be employed to produce
DNA, which can encode proteins for the recombinant synthesis of the CRIg
variants of the invention. For instance, it is possible to derive DNA
based on naturally occurring DNA sequences that encode for changes in an
amino acid sequence of the resultant protein. These mutant DNA can be
used to obtain the CRIg variants of the present invention. These
techniques contemplate, in simplified form, obtaining a gene encoding a
native CRIg polypeptide, modifying the genes by recombinant techniques
such as those discussed below, inserting the genes into an appropriate
expression vector, inserting the vector into an appropriate host cell,
culturing the host cell to cause expression of the desired CRIg variant,
and purifying the molecule produced thereby.

[0118]Somewhat more particularly, a DNA sequence encoding a CRIg variant
of the present invention is obtained by synthetic construction of the DNA
sequence as described in standard textbooks, such as, for example,
Sambrook, J. et al., Molecular Cloning (2nd ed.), Cold Spring Harbor
Laboratory, N.Y., (1989).

[0119]a. Oligonucleotide-Mediated Mutagenesis

[0120]Oligonucleotide-mediated mutagenesis is the preferred method for
preparing substitution, deletion, and insertion variants of a native CRIg
polypeptide or a fragment thereof. This technique is well known in the
art as described by Zoller et al., Nucleic Acids Res. 10: 6487-6504
(1987). Briefly, nucleic acid encoding the starting polypeptide sequence
is altered by hybridizing an oligonucleotide encoding the desired
mutation to a DNA template, where the template is the single-stranded
form of the plasmid containing the unaltered or native DNA sequence of
encoding nucleic acid. After hybridization, a DNA polymerase is used to
synthesize an entire second complementary strand of the template which
will thus incorporate the oligonucleotide primer, and will code for the
selected alteration of starting nucleic acid.

[0121]Generally, oligonucleotides of at least 25 nucleotides in length are
used. An optimal oligonucleotide will have 12 to 15 nucleotides that are
completely complementary to the template on either side of the
nucleotide(s) coding for the mutation. This ensures that the
oligonucleotide will hybridize properly to the single-stranded DNA
template molecule. The oligonucleotides are readily synthesized using
techniques known in the art such as that described by Crea et al., Proc.
Natl. Acad Sci. USA 75: 5765 (1978).

[0122]If phage display is used, the DNA template can only be generated by
those vectors that are either derived from bacteriophage M13 vectors (the
commonly available M13 mp 18 and M13 mp19 vectors are suitable), or those
vectors that contain a single-stranded phage origin or replication as
described by Viera et al., Meth. Enzymol. 153:3 (1987). Thus, the DNA
that is to be mutated must be inserted into one of these vectors in order
to generate a single-stranded template. Production of the single-stranded
template is described in sections 4.21-4.41 of Sambrook et al., supra.

[0123]To alter the native DNA sequence, the oligonucleotide is hybridized
to the single stranded template under suitable hybridization conditions.
A DNA polymerizing enzyme, usually the Klenow fragment of DNA polymerase
I, is then added to synthesize the complementary strand of the template
using the oligonucleotide as a primer for synthesis. A heteroduplex
molecule is thus formed such that one strand of DNA encodes the mutated
form of CRIg, and the other strand (the original template) encodes the
native, unaltered sequence of CRIg. This heteroduplex molecule is then
transformed into a suitable host cell, usually a prokaryote such as E.
coli JM-101. After growing the cells, they are plated onto agarose plates
and screened using the oligonucleotide primer radiolabelled with
32Phosphate to identify the bacterial colonies that contain the
mutated DNA.

[0124]The method described immediately above may be modified such that a
homoduplex molecule is created wherein both strands of the plasmid
contain the mutation(s). The modifications are as follows: The
single-stranded oligonucleotide is annealed to the single-stranded
template as described above. A mixture of three deoxyribonucleotides,
deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and
deoxyribothymidine (dTTP), is combined with a modified
thio-deoxyribocytosine called dCTP-(aS) (Amersham). This mixture is added
to the template-oligonucleotide complex. Upon addition of DNA polymerase
to this mixture, a strand of DNA identical to the template except for the
mutated bases is generated. In addition, this new strand of DNA will
contain dCTP-(aS) instead of dCTP, which serves to protect it from
restriction endonuclease digestion. After the template strand of the
double-stranded heteroduplex is nicked with an appropriate restriction
enzyme, the template strand can be digested with ExoIII nuclease or
another appropriate nuclease past the region that contains the site(s) to
be mutagenized. The reaction is then stopped to leave a molecule that is
only partially single-stranded. A complete double-stranded DNA homoduplex
is then formed using DNA polymerase in the presence of all four
deoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplex
molecule can then be transformed into a suitable host cell such as E.
coli JM101, as described above.

[0125]Mutants with more than one amino acid to be substituted may be
generated in one of several ways. If the amino acids are located close
together in the polypeptide chain, they may be mutated simultaneously
using one oligonucleotide that codes for all of the desired amino acid
substitutions. If, however, the amino acids are located some distance
from each other (separated by more than about ten amino acids), it is
more difficult to generate a single oligonucleotide that encodes all of
the desired changes. Instead, one or two alternative methods may be
employed.

[0126]In the first method, a separate oligonucleotide is generated for
each amino acid to be substituted. The oligonucleotides are then annealed
to the single-stranded template DNA simultaneously, and the second strand
of DNA that is synthesized from the template will encode all of the
desired amino acid substitutions. The alternative method involves two or
more rounds of mutagenesis to produce the desired mutant. The first round
is as described for the single mutants: wild-type DNA is used for the
template, and oligonucleotide encoding the first desired amino acid
substitution(s) is annealed to this template, and the heteroduplex DNA
molecule is then generated. The second round of mutagenesis utilizes the
mutated DNA produced in the first round of mutagenesis as the template.
Thus, this template already contains one or more mutations. The
oligonucleotide encoding the additional desired amino acid
substitution(s) is then annealed to this template, and the resulting
strand of DNA now encodes mutations from both the first and second rounds
of mutagenesis. This resultant DNA can be used as a template in a third
round of mutagenesis, and so on.

[0127]b. Cassette Mutagenesis

[0128]This method is also a preferred method for preparing substitution,
deletion, and insertion variants of CRIg. The method is based on that
described by Wells et al. Gene 34: 315 (1985). The starting material is
the plasmid (or other vector) comprising gene 1, the gene to be mutated.
The codon(s) to be mutated in the nucleic acid encoding the starting CRIg
molecule are identified. There must be a unique restriction endonuclease
site on each side of the identified mutation site(s). If no such
restriction sites exist, they may be generated using the above-described
oligonucleotide-mediated mutagenesis method to introduce them at
appropriate locations in gene 1. After the restriction sites have been
introduced into the plasmid, the plasmid is cut at these sites to
linearize it. A double-stranded oligonucleotide encoding the sequence of
the DNA between the restriction sites but containing the desired
mutation(s) is synthesized using standard procedures. The two strands are
synthesized separately and then hybridized together using standard
techniques. This double-stranded oligonucleotide is referred to as the
cassette. This cassette is designed to have 3' and 5' ends that are
compatible with the ends of the linearized plasmid, such that it can be
directly ligated to the plasmid. This plasmid now contains the mutated
DNA sequence of CRIg.

[0129]c. Recombinant Production of CRIG Variants

[0130]The DNA encoding variants are then inserted into an appropriate
plasmid or vector. The vector is used to transform a host cell. In
general, plasmid vectors containing replication and control sequences
which are derived from species compatible with the host cell are used in
connection with those hosts. The vector ordinarily carries a replication
site, as well as sequences which encode proteins that are capable of
providing phenotypic selection in transformed cells.

[0131]For example, E. Coli may be transformed using pBR322, a plasmid
derived from an E. coli species (Mandel, M. et al., (1970) J. Mol. Biol.
53:154). Plasmid pBR322 contains genes for ampicillin and tetracycline
resistance, and thus provides easy means for selection. Other vectors
include different features such as different promoters, which are often
important in expression. For example, plasmids pKK223-3, pDR720, and
pPL-λ, represent expression vectors with the tac, trp, or PL
promoters that are currently available (Pharmacia Biotechnology).

[0132]Other preferred vectors can be constructed using standard techniques
by combining the relevant traits of the vectors described herein.
Relevant traits of the vector include the promoter, the ribosome binding
site, the variant gene or gene fusion, the signal sequence, the
antibiotic resistance markers, the copy number, and the appropriate
origins of replication.

[0133]Suitable host cells for cloning or expressing the DNA in the vectors
herein include prokaryote, yeast, or higher eukaryote cells. Suitable
prokaryotes for this purpose include eubacteria, such as Gram-negative or
Gram-positive organisms, for example, Enterobacteriaceae such as
Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,
Salmonella, e.g., Salmonella typhimurium, Serrafia, e.g, Serratia
marcescans, and Shigeila, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published Apr. 12, 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC
31,446), although other strains such as E. coli B, E. coli X 1776 (ATCC
31,537), and E coil W31 10 (ATCC 27,325) are suitable. These examples are
illustrative rather than limiting.

[0135]Suitable host cells for the expression of glycosylated antibody are
derived from multicellular organisms. Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains and variants
and corresponding permissive insect host cells from hosts such as
Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes
albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx
mori have been identified. A variety of viral strains for transfection
are publicly available, e.g., the L-1 variant of Autographa califomica
NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used
as the virus herein according to the present invention, particularly for
transfection of Spodoptera frugiperda cells. Plant cell cultures of
cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be
utilized as hosts.

[0137]Host cells are transformed with the above-described expression or
cloning vectors for the production of the CRIg variants herein and
cultured in conventional nutrient media modified as appropriate for
inducing promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.

[0138]The host cells used to produce the CRIg variants of this invention
may be cultured in a variety of media. Commercially available media such
as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640
(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are
suitable for culturing the host cells. In addition, any of the media
described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.
Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;
4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.
30,985 may be used as culture media for the host cells. Any of these
media may be supplemented as necessary with hormones and/or other growth
factors (such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate), buffers
(such as HEPES), nucleotides (such as adenosine and thymidine),
antibiotics (such as GENTAMYCIN®), trace elements (defined as
inorganic compounds usually present at final concentrations in the
micromolar range), and glucose or an equivalent energy source. Any other
necessary supplements may also be included at appropriate concentrations
that would be known to those skilled in the art. The culture conditions,
such as temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the ordinarily
skilled artisan.

[0139]When using recombinant techniques, the CRIg variant can be produced
intracellularly, in the periplasmic space, or directly secreted into the
medium. If the CRIg variant is produced intracellularly, as a first step,
the particulate debris, either host cells or lysed cells, is removed, for
example, by centrifugation or ultrafiltration. Where the CRIg variant is
secreted into the medium, supernatants from such expression systems are
generally first concentrated using a commercially available protein
concentration filter, for example, an Amicon or Millipore Pellicon
ultrafiltration unit. A protease inhibitor such as PMSF may be included
in any of the foregoing steps to inhibit proteolysis and antibiotics may
be included to prevent the growth of adventitious contaminants.

[0142]The CRIg variants of the present invention may also be modified in a
way to form a chimeric molecule comprising CRIg variant, including
fragments thereof, fused to another, heterologous polypeptide or amino
acid sequence. In one embodiment, such a chimeric molecule comprises a
fusion of CRIg variant, or a fragment thereof, with a tag polypeptide
which provides an epitope to which an anti-tag antibody can selectively
bind. The epitope tag is generally placed at the amino- or
carboxyl-terminus of the variant CRIg polypeptide. The presence of such
epitope-tagged forms of the CRIg variant can be detected using an
antibody against the tag polypeptide. Also, provision of the epitope tag
enables the CRIg polypeptide to be readily purified by affinity
purification using an anti-tag antibody or another type of affinity
matrix that binds to the epitope tag. Various tag polypeptides and their
respective antibodies are well known in the art. Examples include
poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags;
the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol.
Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10,
G4, B7 and 9E10 antibodies thereto (Evan et al., Molecular and Cellular
Biology, 5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D
(gD) tag and its antibody (Paborsky et al., Protein Engineering,
3(6):547-553 (1990)). Other tag polypeptides include the Flag-peptide
(Hopp et al., BioTechnology, 6:1204-1210 (1988)); the KT3 epitope peptide
(Martin et al., Science, 255:192-194 (1992)); an quadrature-tubulin
epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)];
and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc.
Natl. Acad. Sci. USA, 87:6393-6397 (1990)).

[0143]In another embodiment, the chimeric molecule may comprise a fusion
of the CRIg variant or a fragment thereof with an immunoglobulin or a
particular region of an immunoglobulin. For a bivalent form of the
chimeric molecule, such a fusion could be to the Fc region of an IgG
molecule. These fusion polypeptides are antibody-like molecules which
combine the binding specificity of a heterologous protein (an "adhesin")
with the effector functions of immunoglobulin constant domains, and are
often referred to as immunoadhesins. Structurally, the immunoadhesins
comprise a fusion of an amino acid sequence with the desired binding
specificity which is other than the antigen recognition and binding site
of an antibody (i.e., is "heterologous"), and an immunoglobulin constant
domain sequence. The adhesin part of an immunoadhesin molecule typically
is a contiguous amino acid sequence comprising at least the binding site
of a receptor or a ligand. The immunoglobulin constant domain sequence in
the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1,
IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,
IgD or IgM.

[0144]The simplest and most straightforward immunoadhesin design combines
the binding region(s) of the "adhesin" protein with the hinge and Fc
regions of an immunoglobulin heavy chain. Ordinarily, when preparing the
CRIg-immunoglobulin chimeras of the present invention, nucleic acid
encoding the extracellular domain of CRIg will be fused C-terminally to
nucleic acid encoding the N-terminus of an immunoglobulin constant domain
sequence, however N-terminal fusions are also possible.

[0145]Typically, in such fusions the encoded chimeric polypeptide will
retain at least functionally active hinge and CH2 and CH3 domains of the
constant region of an immunoglobulin heavy chain. Fusions are also made
to the C-terminus of the Fc portion of a constant domain, or immediately
N-terminal to the CH1 of the heavy chain or the corresponding region of
the light chain.

[0146]The precise site at which the fusion is made is not critical;
particular sites are well known and may be selected in order to optimize
the biological activity, secretion or binding characteristics of the
CRIg-immunoglobulin chimeras.

[0147]In some embodiments, the CRIg-immunoglobulin chimeras are assembled
as monomers, or hetero- or homo-multimer, and particularly as dimers or
tetramers, essentially as illustrated in WO 91/08298.

[0148]In a preferred embodiment, the CRIg extracellular domain sequence is
fused to the N-terminus of the C-terminal portion of an antibody (in
particular the Fc domain), containing the effector functions of an
immunoglobulin, e.g. immunoglobulin G.sub. 1 (IgG 1). It is possible to
fuse the entire heavy chain constant region to the CRIg extracellular
domain sequence. However, more preferably, a sequence beginning in the
hinge region just upstream of the papain cleavage site (which defines IgG
Fc chemically; residue 216, taking the first residue of heavy chain
constant region to be 114, or analogous sites of other immunoglobulins)
is used in the fusion. In a particularly preferred embodiment, the CRIg
amino acid sequence is fused to the hinge region and CH2 and CH3, or to
the CH1, hinge, CH2 and CH3 domains of an IgG 1, gG2, or IgG3 heavy
chain. The precise site at which the fusion is made is not critical, and
the optimal site can be determined by routine experimentation.

[0149]In some embodiments, the CRIg-immunoglobulin chimeras are assembled
as multimer, and particularly as homo-dimers or -tetramers. Generally,
these assembled immunoglobulins will have known unit structures. A basic
four chain structural unit is the form in which IgG, IgD, and IgE exist.
A four unit is repeated in the higher molecular weight immunoglobulins;
IgM generally exists as a pentamer of basic four units held together by
disulfide bonds. IgA globulin, and occasionally IgG globulin, may also
exist in multimeric form in serum. In the case of multimer, each four
unit may be the same or different.

[0150]Alternatively, the CRIg extracellular domain sequence can be
inserted between immunoglobulin heavy chain and light chain sequences
such that an immunoglobulin comprising a chimeric heavy chain is
obtained. In this embodiment, the CRIg sequence is fused to the 3' end of
an immunoglobulin heavy chain in each arm of an immunoglobulin, either
between the hinge and the CH2 domain, or between the CH2 and CH3 domains.
Similar constructs have been reported by Hoogenboom et al., Mol.
Immunol., 28:1027-1037 (1991).

[0151]Although the presence of an immunoglobulin light chain is not
required in the immunoadhesins of the present invention, an
immunoglobulin light chain might be present either covalently associated
to a CRIg-immunoglobulin heavy chain fusion polypeptide, or directly
fused to the CRIg extracellular domain. In the former case, DNA encoding
an immunoglobulin light chain is typically coexpressed with the DNA
encoding the CRIg-immunoglobulin heavy chain fusion protein. Upon
secretion, the hybrid heavy chain and the light chain will be covalently
associated to provide an immunoglobulin-like structure comprising two
disulfide-linked immunoglobulin heavy chain-light chain pairs. Methods
suitable for the preparation of such structures are, for example,
disclosed in U.S. Pat. No. 4,816,567 issued Mar. 28, 1989.

[0152]Pharmaceutical Compositions

[0153]The CRIg variants of the present invention can be administered for
the treatment of diseases the pathology of which involves the alternative
complement pathway.

[0154]Therapeutic formulations are prepared for storage by mixing the
active molecule having the desired degree of purity with optional
pharmaceutically acceptable carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]),
in the form of lyophilized formulations or aqueous solutions. Acceptable
carriers, excipients, or stabilizers are nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid and methionine; preservatives (such as
octadecyldirnethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl
alcohol; alkyl parabens such as methyl or propyl paraben; catechol;
resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight
(less than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine,
histidine, arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating agents
such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.
Zn-protein complexes); and/or non-ionic surfactants such as TWEEN®,
PLURONICS® or polyethylene glycol (PEG).

[0155]Lipofections or liposomes can also be used to deliver the
polypeptide, antibody, or an antibody fragment, into cells. Where
antibody fragments are used, the smallest fragment which specifically
binds to the binding domain of the target protein is preferred. For
example, based upon the variable region sequences of an antibody, peptide
molecules can be designed which retain the ability to bind the target
protein sequence. Such peptides can be synthesized chemically and/or
produced by recombinant DNA technology (see, e.g. Marasco et al., Proc.
Nati. Acad. Sci. USA 90, 7889-7893 [1993]).

[0156]The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not adversely
affect each other. Such molecules are suitably present in combination in
amounts that are effective for the purpose intended.

[0157]The active molecules may also be entrapped in microcapsules
prepared, for example, by coascervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example, liposomes,
albumin microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0158]The formulations to be used for in vivo administration must be
sterile. This is readily accomplished by filtration through sterile
filtration membranes.

[0159]Sustained-release preparations may be prepared. Suitable examples of
sustained-release preparations include semipermeable matrices of solid
hydrophobic polymers containing the antibody, which matrices are in the
form of shaped articles, e.g. films, or microcapsules. Examples of
sustained-release matrices include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides
(U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and gamma.
ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable
lactic acid-glycolic acid copolymers such as the LUPRON DEPOT®
(injectable microspheres composed of lactic acid-glycolic acid copolymer
and leuprolide acetate), and poly-D-(--)-3-hydroxybutyric acid. While
polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid
enable release of molecules for over 100 days, certain hydrogels release
proteins for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a result of
exposure to moisture at 37 C, resulting in a loss of biological activity
and possible changes in immunogenicity. Rational strategies can be
devised for stabilization depending on the mechanism involved. For
example, if the aggregation mechanism is discovered to be intermolecular
S--S bond formation through thio-disulfide interchange, stabilization may
be achieved by modifying sulfhydryl residues, lyophilizing from acidic
solutions, controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.

[0160]Methods of Treatment

[0161]As a result of their ability to inhibit complement activation, in
particular the alternative complement pathway, the CRIg variants of the
present invention find utility in the prevention and/or treatment of
complement-associated diseases and pathological conditions. Such diseases
and conditions include, without limitation, complement-associated,
inflammatory and autoimmune diseases.

[0162]Specific examples of complement-associated, inflammatory and immune
related diseases and disorders that can be targeted by the CRIg variants
herein have been listed earlier.

[0163]Further details of the invention are illustrated by the following
non-limiting Examples.

[0170]Side-directed mutagenesis for the point mutations was carried out as
above by using appropriated codons to produce the respective mutations,
and the correct clones were confirmed by sequence.

[0171]Library sorting and screening to select CRIg variants:

[0172]Maxisorp immunoplates were coated overnight at 4° C. with C3b
(5 μg/ml) and blocked for 1 hr at room temperature with
phosphate-buffered saline (PBS) and 0.05% (w/v) bovine serum albumin
(BSA). Phage libraries were added to the C3b coated plates and incubated
at room temperature for 3 hr. The plates were washed ten times and bound
phage were eluted with 50 mM HCl and neutralized with equal volume of 1.0
M Tris base (pH7.5). Recovered phages were amplified by passage through
E. coli XL 1-blue and were used for additional rounds of binding
selections. After 5 rounds, we select 12 individual clones from each
library and grow them in a 96-well format in 500 μl of 2YT broth
supplemented with carbenicillin and M13-KO7 helper phage. Two-fold serial
diluted culture supernatants were added directly in 384 well plates by
coated with C3b, anti-gD, BSA and unrelated protein as designed
positions. Binding affinity was measured to estimate a phage
concentration giving C3b significant higher than anti-gD but not to BSA
and unrelated protein. We fixed the phage concentration, screening about
200 clones from each library in the same format and selected 24-48 clones
in which showed significantly to bind to C3b over anti-gD from each
library, then sequence them for analysis.

[0173]Competitive Phage ELISA

[0174]For estimating the binding affinity, a modified phage ELISA was
used. The 96 well microtiter plates were coat with 2 ug/ml 3Cb in 50 mM
carbonate buffer (pH9.6) at 4C over- night. The plates were then block
with PBS, 0.5% BSA for 1 hour at room temperature. Phage displaying CRIg
variants serially diluted in PBT buffer and binding was measured to
estimate a phage concentration giving 50% of the signal at saturation.
Subsaturating concentration of phage was fixed and pre-incubated for 2 h
with serial dilutions of C3b, then transferred the mixture to assay
plates coated with C3b. After incubating 15 min, the plates were washed
with PBS, 0.05% Tween 20 and incubated 30 min with horseradish
peroxidase/anti-M13 antibody conjugate (1:5000 dilution in PBT buffer).
The plates were washed, developed with TMB substrate, quenched with 1.0 M
H3PO4, and read spectrophotometrically at 450 nm. The affinity
(Ic50) was calculated as the concentration of competing C3b that resulted
in half-maximal phagemid binding.

[0175]Protein Purification

[0176]A single colony of E. coli. BL21(DE3) harboring the expression
plasmid was inoculated into 30 mL of LB medium supplemented with 50
μg/mL carbenicillin (LB/carb medium) and was grown overnight at
37° C. The bacteria were harvested, washed, resuspended, and
inoculated into 500 mL of LB/carb medium. The culture was grown at
37° C. to mid-log phase (A600=0.8). Protein expression was
induced with 0.4 mM isopropyl 1-thio-D-galactopyranoside, and the culture
was grown for 24 h at 30° C. The bacteria were pelleted by
centrifugation at 4000 g for 15 min, washed twice with phosphate-buffered
saline (PBS), and frozen for 8 h at -80° C. The pellet was
resuspended in 50 mL of PBS, and the bacteria were lysed by passing
through the Microfluidizer Processing or sonicate equipments. The CRIg
variant proteins were purified with 2 ml NI-NTA agarose and gel
filtration.

[0177]mutCRIg-huFc Fluid Phase Competitive Binding ELISA:

[0178]huCRIg(L)-LFH was diluted to 2 ug/mL in PBS, pH 7.4, and coated onto
Maxisorp 384-well flat bottom plates (Nunc, Neptune, N.J.) by incubating
overnight (16-18 hr) at 4° C. (25 ul/well). The plates were washed
3 times in Wash Buffer (PBS, pH7.4, 0.05% Tween 20), and 50 ul/well of
Block Buffer (PBS, pH 7.4, 0.5% BSA) was added to each well. The plates
were allowed to block for 1-3 hr; this and all subsequent incubations
were performed on an orbital shaker at room temperature. During the
blocking step, C3b (purified at Genentech) was diluted to 2OnM in Assay
Buffer (PBS pH7.4, 0.5% BSA, 0.05% Tween-20), and the mutCRIg-huFc
molecules were serially diluted in Assay Buffer, over a concentration
range of 20,000-0.34 nM. The C3b and mutCRIg-huFc molecules were then
mixed 1:1 and allowed to pre-incubate for 0.5-1 hr. The blocked plates
were washed three times (as described above), and the C3b:mutCRIg-huFc
complexes were added to the reaction plates (25 ul/well). After a 1-2hr
incubation, The ELISA plates were washed three times, (as described
above) and plate-bound C3b was detected by the addition of an anti-human
C3b antibody (clone 5F202, US Biological, Swampscott, Mass.; 600 ng/mL,
25 ul/well). The plates were incubated for 1-2hr and washed as described
above. HRP-conjugated anti-murine Fc IgG (Jackson ImmunoResearch, West
Grove, PA) diluted 1:2,000 was then added (25 ul/well), and the plates
were incubated for 1-2 hr. After a final wash, 25 ul/well of TMB
substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) was added
to the ELISA plates. Color development was stopped after approximately 8
min by adding 25 ul/well 1.0M phosphoric acid. Absorbance at 450 nm and
650 nm was determined using a SpectraMax 250 microtiter plate reader
(Molecular Devices, Sunnyvale, Calif.).

[0179]Complement Activation Assay:

[0180]The ability of mutCRIg-Fc to inhibit complement activation was
evaluated using the WieslabTM Complement System Alternative Pathway Kit
(Alpco Diagnostics, Salem, N.H.). Serially diluted mutCRIg-Fc (400 to 0.2
nM) and Clq deficient human serum (5%) (Complement Technology, Tyler, TX)
were prepared at twice the final desired concentration, mixed 1:1, and
pre-incubated for 5min on an orbital shaker at 300 RPM prior to adding to
the LPS-coated ELISA plates (100 ul/well). The remainder of the assay was
following manufacturer's instructions. Briefly, the samples in the ELISA
plates were incubated for 60-70 min at 37° C. and then washed
three times in Wash Buffer (PBS, pH7.4, 0.05% Tween 20). 100 ul/well of
the anti-C5b-9 conjugate was added to the ELISA plate. After a 30 min
incubation at room temperature, the ELISA plate was washed as described
above, and 100 ul of substrate was added per well, and the plates were
incubated at room temperature for an additional 30 min. The color
development was stopped by adding 50 ul/well of 5 mM EDTA. Absorbance at
405 nm was determined using a MultiSkan Ascent microtiter plate reader
(Thermo Fisher Scientific, Milford, Mass.).

[0181]Hemolysis Inhibition Assay:

[0182]Rabbit red blood cells (Colorado Serum Company, Denver, Colo.) were
washed three times with Veronal Buffer (Sigma, St. Louis, MO) containing
0.1% bovine skin gelatin (Sigma) (GVB), centrifuging at 1500 rpm,
4° C. for 10 minutes for each wash. After the final centrifugation
step, the cells were resuspended in GVB at a final concentration of
2×109 cells/mL. Complement inhibitors serially diluted in GVB
were added to 96-well U-bottom polypropylene plate(s) (Costar, Cambridge,
Mass.) at 50 μL/well followed by 20 μL/well of rabbit red blood
cells diluted 1:2 in 0.1 M MgCl2bl /0.1 M EGTA/GVB. The in-plate
complement cascade was triggered by the addition of 30 μL/well
C1q-depleted serum (Complement Technology, Tyler, Tex.), pre-diluted 1:3
with GVB. The plate(s) were incubated with gentle agitation for 30
minutes at room temperature before stopping the reaction with 100
μL/well 10 mM EDTA/GVB. After centrifuging the plate(s) at 1500 rpm
for 5 minutes, the supernatants were transferred to clear flat bottom,
non-binding, 96-well plate(s) (Nunc, Neptune, N.J.) and the optical
densities were read at 412 nm using a microplate reader (Molecular
Devices, Sunnyvale, Calif.).

[0183]Alpha Screen Competitive Assay:

[0184]The potential cross-reacivity of the mutant CRIg molecules to C3 was
evaluated using the AlphaScreen® Histidine (Nickel Chelate) Detection
Kit (PerkinElmer, Waltham, Mass.). Serially diluted human C3 and C3b
(3,000 to 0.7 nM), as well as fixed concentrations of biotinylated iC3b
(30 nM), and both mutant CRIg (mutCRIg) and wild-type CRIg molecules
(15-60 nM) were prepared at three times the final desired concentration,
mixed 1:1:1, and pre-incubated at ambient temperature for 30 minutes on
an orbital shaker at 3000 TPM. A 1:1 mixture of streptavidin donor beads
and nickel chelate acceptor beads (0.1 mg/mL each) was prepared at four
times the final desired concentration and added to the reaction. The
reaction plate was incubated at ambient temperature for 60 minutes on an
orbital shaker at 3000 rpm protected from light. The plate was analyzed
on an AlphaQuest®-HTS microplate analyzer (PerkinElmer, Waltham,
Mass.).

[0185]Surface Plasmon Resonance

[0186]Affinities of C3b for mutant and wild-type CRIg were determined by
using surface plasmon resonance measurements on a Biacore® A100
instrument (GE healthcare). An anti-Fc capture format was employed and
the KD was calculated from equilibrium binding measurements. The sensor
chip was prepared using the anti-muFc capture kit (BR-1008-38) following
instructions supplied by the manufacturer. Mutant or wild-type CRIg was
diluted in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.01%
Tween-20) to 1 μg/mL and injections of 60 μL were made such that
˜100 RU of fusion protein were captured on one spot of the chip
surface. Sensorgrams were recorded for 10 min injections of solutions of
varied C3b concentration over the CRIg spot with subtraction of signal
for a reference spot containing the capture antibody but no CRIg. Data
were obtained for a 2-fold dilution series of C3b ranging in
concentration from 4 μM to 15.6 nM with the flow rate at 10 μL/min
and at a temperature of 25° C. The surface was regenerated between
binding cycles by a 30 second injection of 10 mM Gly-HCl pH 1.7. Plateau
values obtained at the end of each C3b injection were used to calculate
KD using the Affinity algorithm of the Biacore A100 Evaluation
Software v1.1 (Safsten et al. (2006) Anal. Biochem. 353:181).

[0187]Results

[0188]Phage Library Design

[0189]We used the crystal structure of CRIg in complex with C3b to design
target libraries. Five libraries were designed to cover the contact area
between CRIg and C3b (FIG. 4). CRIg libraries were constructed as a
fusion to the g3p minor coat protein in a monovalent phage display vector
(Zhang et al., J Biol Chem 281(31): 22299-311 (2006)). We introduced stop
codons by mutagenesis into the CRIg-coding portion of the phage plasmid
at each residue to be randomized. Each construct containing a stop-codon
was then used to generate the phage-display library (see material and
method). A "soft randomization" strategy was used to select binders to
maintain a wild-type sequence bias such that the selected positions were
mutated only 50% of the time. All five libraries were obtained with an
average diversity of>1010 independent sequences per library.
(Table 1).

[0190]Selections with CRIg Phage Library

[0191]Following four rounds of binding selection, we obtained 38 unique
clones from thes five libraries. (Table 2). In library 1, lysine at
position 15 was conserved. Aromatic residues, tyrosine and tryptophan,
replaced glutamic acid at position 8. Position 14 was occupied by either
the parental tryptophan or a homologous phenylalanine. In library 2, we
sequenced 24 clones and all of them revealed consensus. Position 42, 46
and 47 were conserved as wild type. Asparagin, histidine and
phenylalanine replaced the wild type sequence at position 43, 44 and 45.
In library 3, we randomized 10 positions and the sequences exhibited
complete conservation at position 54, 55, 56, 57, 58, 61, 62 and 63.
Isoleucine or lysine was occupied at position 60. At position 64,
glutamine was replaced by arginine or conserved. In library 4, aromatic
residues dominated at position 86 and homologous basic residues, arginine
and lysine, dominated at position 99. Position 85, 87 and 95 were also
soft randomized, but appeared highly conserved. In library 5, two
significant homologous basic residues, lysine and arginine were preferred
over glutamine at position 105. Negatively charged residues, aspartic
acid or acidic residues, asparagine was dominated at position 110.

[0192]We estimated the affinities of some of the mutants by competitive
phage ELISA (data not shown), and we found that there were clones in
library 3 which were approximately eightfold times better C3b binders
than wild type CRIg.

[0193]Determination of In Vitro Binding Affinity and In Vivo Biological
Potency

[0194]In order to identify critical residues for increasing the binding
affinity to C3b and potency in hemolytic inhibition assay, the next
approach was to design second generation of CRIg variants by
incorporating dominant single mutation and keep other positions as wild
type, or choosing 2-3 high-affinity clones from first generation
phage-libraries which were determined by phage ELISA. In order to
accurately measure the affinity and potency of our mutants, we expressed
all the variants as isolated proteins. The results (Table 3) from
hemolytic inhibition assay showed that L12 from library 1, L33 from
library 3 and L41 from library 4 significantly increased the potency by 4
to 10 fold compare to wild type in a hemolytic assay. L32 from library 3
showed a 10 fold improved IC50 compared to wild type CRIg. The data also
demonstrated that the binding affinity and the potency from the
cell-based assays were not correlated.

[0195]Combination of Mutants

[0196]Based on the results from the second generation labraries, we
designed the third generation of mutants in order to further improve the
potency in the hemolytic assay and binding affinity. We chose three of
the biologically most potent mutants (L12-8W, L33-Q60I/L32-Q64R and
L41-M86Y) and one of the highest binding affinity mutant (L32-Q64R) as a
template. Then we combined these mutants with other biological potent
clones obtained in the second generation of libraries to determine an
optimal set of mutations that increase potency in the hemolytic assay and
binding affinity. We expressed and purified the CRIg variants for
detailed analysis. The data showed (Table 4) that the combo mutants from
L12 didn't improve inhibition potency and even displayed a lower activity
compare with the parent mutant despite a 3-6 fold higher binding affinity
of the WL41 and WL59 mutants. Within the mutants from L32, RL41
demonstrated a 1.8 fold better binding affinity than wild type and a 6
fold better potency in the hemolytic assay. All the mutants from L33
group showed the significant increased binding affinity; about a 27-226
fold increase compared with wild type although the potency in the
hemolysis assay did not increase significantly. We also noticed that
60I-64R and 86Y was involved in most the affinity improved combo clones.

[0198]We selected mutant Q64R M86Y, which had the highest affinity in the
competitive ELISA (FIG. 4 and Table 3) for further analysis. In order to
determine the binding affinity of CRIg wt and CRIg Q64R M86Y for C3b,
Biacore analysis of CRIg wt and CRIg Q64R M86Y was performed. The
affinity of CRIg Q64R M86Y was improved 5 fold over wildtype CRIg (FIG.
6). Previous studies have shown that CRIg wt selectively binds to C3b but
not to native C3 (Wiesman et al., Nature, 444(7116):217-20, 2006). Since
mutagenesis may change this selectivity we compared the affinity of CRIg
Q64R M86Y for C3b versus C3 in an alpha-screen fluid-phase competitive
assay. CRIg Q64R M86Y competed with soluble C3b, but not with soluble C3,
indicating that mutagenesis did not affect the selectivity of CRIg for
the active component C3b (FIG. 7). This selectivity was further confirmed
by analysis of these residues in the structure of CRIg Q64R M86Y in
complex with C3b (data not shown).

[0199]To test whether the improved affinity and conserved selectivity for
C3b translates into improved efficacy, we tested CRIg Q64R M86Y versus
CRIg wt in an erythrocyte-based hemolytic assay selective for the
alternative pathway of complement. CRIg Q64R M86Y showed a 4-fold
improved IC50 as compared to CRIg wt (FIG. 8A). To further substantiate
improved potency toward alternative pathway complement inhibiton, we
compared inhibitory activity of CRIg Q64R M86Y with CRIg wt in a
LPS-based assay selective for the alternative pathway of complement.
Here, CRIg showed a 180-fold improvement in IC50 as compared to the
wildtype recombinant protein. CRIg wt and CRIg Q64R M86Y did not affect
complement activation through the classical pathway. Thus, a two amino
acid substitution in the CRIg-C3b binding interface results in a molecule
with improved binding affinity and superior complement inhibitory
activity in two different assays with selectivity for the alternative
pathway of complement.

[0201]Here, CRIg efficacy was tested in a third preclinical model of
immune complex-mediated arthritis. A spontaneous murine model of
rheumatoid arthritis, K/BxN, mimics many of the clinical and histologic
features of human disease with arthritis. Mice were injected with 50
microliter serum obtained from K/B×N mice on day 0. Animals were
checked daily and the extent of disease was scored by visual observation.
All mice were sacrificed on day 6.

[0202]Mice were injected subcutaneously with indicated amount of either
isotype control or hCRIg-mIgGl or hCRIg-RL41-mIgGl recombinant proteins
daily in 100 ul sterile saline starting on day -1.

[0203]Monitoring and Scoring:

[0204]Score for each paw.

[0205]0=No evidence of erythema and swelling

[0206]1=Erythema and mild swelling confined to the mid-foot (tarsal) or
ankle

[0207]2=Erythema and mild swelling extending from the ankle to the
mid-foot

[0208]3=Erythema and moderate swelling extending from the ankle to the
metatarsal joints

[0209]4=Erythema and severe swelling encompass the ankle, foot and digits

[0212]On day 6, blood sample were collected by intracardiac puncture under
anesthesia before sacrifice. The amount of hCRIg-Fc fusion proteins will
be measured using the serum. Joints were collected for histology
evaluation.

[0214]All patent and literature references cited in the present
specification are hereby expressly incorporated by reference in their
entirety.

[0215]While the present invention has been described with reference to
what are considered to be the specific embodiments, it is to be
understood that the invention is not limited to such embodiments. To the
contrary, the invention is intended to cover various modifications and
equivalents included within the spirit and scope of the appended claims.